IgG2 subclass deficiency: IgG subclass assays and IgG2 concentrations among 8015 blood donors

Among the four IgG subclasses in humans, IgG2 is preferentially expressed in antibodies to carbohydrate antigens whereas IgG1 subclass is commonly associated with antibodies to protein antigens. Because of this association with carbohydrate antigens, values for IgG2 in serum are often used as an index of immunocompetence against carbohydrate antigens. To investigate the value of IgG2 measurements in a general population, we have developed a convenient IgG subclass assay, using monoclonal antibodies and particle concentration fluorescence immunoassay. Our assay is specific, precise, convenient, and accurate. When IgG2 concentrations were determined in the serum samples from 8015 adult blood donors, there were more individuals with low IgG2 concentrations than predicted by the log-normal distribution. The observed distribution suggested the presence of a subpopulation with low IgG2 concentration. Because apparently healthy individuals in a general population have low IgG2 concentrations, IgG2 measurements alone may have a limited clinical usefulness as an index of immune function against carbohydrate antigens.     

84例自身免疫性溶血性贫血IgG抗体亚型与临床意义的分析

目的　研究温抗体型自身免疫性溶血性贫血ＩｇＧ亚型及其临床意义。方法　回顾性分析８４ 例患者ＩｇＧ亚型与临床特点的关系。结果　８４ 例患者中ＩｇＧ１ ＋ＩｇＧ３ ＋ Ｃ３ｄ 型占４５．０％ ,ＩｇＧ１ ＋ＩｇＧ３型占１５ ．５％ ,ＩｇＧ１ ＋ Ｃ３ｄ型占１１．０％ ,Ｃ３ｄ型占１０ ．７％ ,ＩｇＧ１ 型占９．５％ ,ＩｇＧ３ ＋ Ｃ３ｄ占８．３ ％ 。各型中均以女性为主,合计占６９ ．５％ 。ＩｇＧ３ 阳性各型的临床表现相似,各项临床指标异常最严重;ＩｇＧ１ 阳性的各型次之;Ｃ３ｄ 型最轻。Ｈｂ＜６０ ｇ／Ｌ、总胆红素＞ ４０ μｍｏｌ／Ｌ、ＦＨｂ＞ ６０ ｍｇ／Ｌ的患者百分率三组间比较,差异有显著性（Ｐ＜０ ．０５） 。ＩｇＧ１ 型与ＩｇＧ３ 型都随Ｃｏｏｍｂｓ 试验积分增高,其临床表现及溶血程度加重。ＩｇＧ３ 阳性的患者治疗有效率为６８ ．２％ ,ＩｇＧ１ 阳性患者有效率为１００％ 。结论　ＩｇＧ 亚型中以ＩｇＧ１ ＋ＩｇＧ３ 为主。ＩｇＧ３ 阳性的患者临床表现及溶血程度严重,治疗效果差     

Nephelometry of human IgG subclass concentration in serum

An immunoassay method for determination of human IgG subclass concentration by rate nephelometry was developed by using the Beckman Immunochemistry Analyzer, subclass-specific antisera, and a human serum standard. Twelve sera derived from normal as well as gammapathological patients were analyzed. The total IgG concentration, as determined by using a readily available kit, correlated with the sum of the concentration of the individual subclasses. Most of the gammapathological cases were shown to be the IgG1 subclass, which was confirmed by radial immunodiffusion.     

IgG subclass concentrations in sera from 200 normal adults and IgG subclass determination of 106 myeloma proteins: an interlaboratory study

The IgG subclass protein concentrations in sera from 200 normal subjects were determined independently in two laboratories, using the same technique (radial immunodiffusion), the same subclass-specific antibodies and the same calibrator. The coefficients of correlation (rs) between IgG subclass concentrations determined in the two laboratories were 0.487, 0.883, 0.928 and 0.926 for IgG1, IgG2, IgG3 and IgG4, respectively (p less than 0.0005 in all four cases). By the chi-square test for goodness of fit, the frequency distributions observed in the two laboratories were found to differ significantly for all four subclasses (p less than 0.01). In one laboratory, the distributions of IgG1 and IgG2 were not significantly different from normal distributions, whereas the distributions of IgG3 and IgG4 deviated significantly. In the other laboratory, all four subclass distributions were significantly different from normal distributions. In the first laboratory, the IgG2 concentrations were log-normal distributed, whereas in the second, IgG1, IgG2 and IgG4 concentrations were log-normal distributed. We conclude that use of identical reagents does not ensure identical frequency distributions. This finding emphasizes the need for standardization of the measurement technique too. Furthermore, we argue that at present, intralaboratory reference intervals for IgG subclass protein concentrations are necessary. The reference intervals should be based on non-parametric statistics. The subclass of 106 monoclonal IgG proteins, which were demonstrated by agarose gel electrophoresis, was identified by RID for 89 samples. The subclass of the remaining 16 M-components was readily determined by qualitative immunoelectrophoretic analysis using the same subclass-specific antibodies.     

IgG4 subclass antibodies impair antitumor immunity in melanoma

Host-induced antibodies and their contributions to cancer inflammation are largely unexplored. IgG4 subclass antibodies are present in IL-10–driven Th2 immune responses in some inflammatory conditions. Since Th2-biased inflammation is a hallmark of tumor microenvironments, we investigated the presence and functional implications of IgG4 in malignant melanoma. Consistent with Th2 inflammation, CD22⁺ B cells and IgG4⁺-infiltrating cells accumulated in tumors, and IL-10, IL-4, and tumor-reactive IgG4 were expressed in situ. When compared with B cells from patient lymph nodes and blood, tumor-associated B cells were polarized to produce IgG4. Secreted B cells increased VEGF and IgG4, and tumor cells enhanced IL-10 secretion in cocultures. Unlike IgG1, an engineered tumor antigen-specific IgG4 was ineffective in triggering effector cell–mediated tumor killing in vitro. Antigen-specific and nonspecific IgG4 inhibited IgG1-mediated tumoricidal functions. IgG4 blockade was mediated through reduction of FcγRI activation. Additionally, IgG4 significantly impaired the potency of tumoricidal IgG1 in a human melanoma xenograft mouse model. Furthermore, serum IgG4 was inversely correlated with patient survival. These findings suggest that IgG4 promoted by tumor-induced Th2-biased inflammation may restrict effector cell functions against tumors, providing a previously unexplored aspect of tumor-induced immune escape and a basis for biomarker development and patient-specific therapeutic approaches.     

IgE and IgG4 subclass in atopic families.

Raised levels of IgG4 were present in twelve (35%) of thirty-four asthmatic children and raised levels of IgE in twenty-three (68%). Eighteen of 104 first degree relatives also had raised levels of IgG4, thirteen had no history of atopic disease and nine failed to give positive skin reactions to Dermatophagoides pteronyssinus or to mixed grass pollens. Fifteen relatives had raised IgE, five without symptoms. No relationship was noted between either raised levels of IgE or IgG4 and infant feeding. Although these immunoglobulin patterns were not consistently associated with symptoms they did tend to be associated in atopic families and it is suggested that the same polygenic factors may govern the IgE and IgG4 levels.     

Evaluation by enzyme-linked immunosorbent assay of IgG anti-D and IgG subclass concentrations in immunoglobulin preparations

BACKGROUND: Anti-D immunoglobulin preparations are injected to prevent hemolytic disease of the newborn. The concentration of IgG anti-D in these preparations is usually determined by an automated hemagglutination technique using as a reference a calibrated preparation of anti-D, but the method requires special equipment and cannot be routinely applied to measure the IgG subclasses of anti-D in these preparations. STUDY DESIGN AND METHODS:Taking advantage of a recently described enzyme-linked immunosorbent assay (ELISA) for the determination of the anti-D concentration in sera of alloimmunized pregnant women, IgG anti-D and IgG subclass concentrations were measured in the international reference preparation (IRP) coded 68/419, 10 anti-D immunoglobulin preparations, and sera of 15 D-immunized volunteers. RESULTS: An IgG anti-D concentration of 61.5 +/- 4.8 microg per ampoule (mean +/- SD) was found by ELISA in IRP 68/419.This result was in agreement with previous determinations obtained by radioimmunoassay (60 microg/ampoule). The IgG subclass concentration of anti-D in this preparation was 48.4 microg of IgG1 (78.6%), 3.0 microg of IgG2 (4.8%), 9.7 microg of IgG3 (15.8%), and 0.4 microg of IgG4 (0.7%). The mean proportion of IgG subclasses of anti-D in 10 immunoglobulin preparations was similar (81.7% for IgG1, 5.0% for IgG2, 12.7% for IgG3, and 0.6% for IgG4). In the sera of 15 immunized volunteers, the IgG anti-D concentration varied from 3.1 to 68.4 microg per mL. The mean IgG subclass composition of anti-D was 79.3 percent for IgG1, 2.2 percent for IgG2, 18.1 percent for IgG3, and 0.4 percent for IgG4. The proportions of IgG3 anti-D in these sera were found to range between 1 percent and 87 percent, as in the sera of D-alloimmunized pregnant women. CONCLUSION: ELISA provides an alternative to the radioimmunoassay and the automated hemagglutination technique. In addition, it allows the evaluation of the absolute concentration of each IgG subclass of anti-D in immunoglobulin preparations and necessitates only the conventional equipment required for an immunoenzymatic assay.     

Immunoblot analysis of IgM and IgG subclass responses in murine toxoplasmosis

The diagnosis of toxoplasmosis still relies heavily on serological methods, particularly the dye test. In order to further elucidate the role of humoral immunity and to distinguish parasite antigens which might be used in new, more specific diagnostic tests, the antibody response in murine toxoplasmosis was examined. Specific antibody was measured by the dye test in sequential serum samples collected at intervals from 3 to 98 days post inoculation. The peptides involved in the immune response were characterised by probing blots with IgM and the four murine IgG isotypes. Antibody was detectable by day 10 and rose to a titre of ≤ 16,000 by day 28. Immunoblotting revealed multiple bands on both IgM and IgG blots as early as day 3. Six intense bands with approximate molecular weights of 80, 67, 43, 35, 30 and 28 kDa developed early and persisted throught the course of infection. Several of these were thought to be surface antigens of the parasite. The initial and strongest IgG response by immunoblotting was seen with the IgG2b isotype, which is believed to play an important role in antibody-dependent cell-mediated cytotoxicity.     

IgG heavy-chain subclass restriction of thyrotropin-binding inhibitory immunoglobulins in Graves'' disease

The IgG subclass composition of antibodies is an important determinant of their function. Thyrotropin receptor antibodies cause the hyperthyroidism of Graves' disease but their subclass distribution has been incompletely investigated. We have therefore purified IgG subclasses from Graves' sera by passage over affinity columns designed to deplete all but a single subclass, and then assayed those pure subclass fractions for their ability to displace radiolabelled thyrotropin from its solubilized receptor as a measure of thyrotropin receptor antibody activity. Sufficient activity was recovered for analysis in nine of 10 Graves' patients, in five of whom activity was almost completely (97-100%) restricted to the IgG1 subclass; in the remaining four patients the response was predominantly IgG1 and IgG4 with marked under-representation of the IgG2 subclass. This contrasts with the unrestricted subclass response, in the same fractions, for autoantibodies against thyroglobulin and microsomes. These results suggest that there may be a primary defect at the B-cell level in Graves' disease.     

Hemagglutination in capillaries: correlation with blood group specificity and IgG subclass

Two hundred four examples of blood group antibodies, reactive only by the indirect antiglobulin test, were examined by the two-layer albumin capillary (TLAC) technique. Fifty-nine per cent of the examples tested were reactive by this technique. TLAC reactivity appeared to be related to antibody specificity, but a positive association between TLAC reactivity and IgG subclass composition was not confirmed.     

Application of clinical laboratory measurements to issues of environmental health.

Monitoring of biochemical constituents in serum is an important component in revealing potential toxicity in humans and experimental animals due to exposure to a variety of xenobiotic agents. The relative toxicity of pure compounds, usually at large doses, has helped elucidate the mode of action of these compounds and their relative risk. However, most actual cases of environmental exposure present an extensive range of components and the potential for synergistic or inhibitory interactions. In this paper we review two such environmental cases: The Love Canal chemical dump site in Niagara Falls, NY, and the transformer fire at the State Office Building in Binghamton, NY. We focus on the clinical laboratory measurements obtained in these studies (including serum glucose, triglycerides, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, lactate dehydrogenase, sodium and potassium), their usefulness, limitations, and application to such cases. Significant alterations in serum triglyceride and alanine aminotransferase levels were found in guinea pigs due to exposure to dioxins. These two tests were useful in estimating the 'equivalent' concentration of 2,3,7,8-tetrachlorodibenzo-p-dioxin in complex chemical mixtures.     

Standardization of IgG subclass antiserums for use with sensitized red cells

The only IgG subclass antiserums standardized for use with red cells are those sold by the Netherlands Red Cross. Their recommended method is a sedimentation antiglobulin test (AGT) not generally used in the USA. Some years ago we tested the antiserums for sensitivity and specificity using the routine centrifugation AGT and found the methods comparable. More recently, we have become concerned since the antiserums are being used by a variety of methods other than the recommended procedure. It is well-known that it is difficult to prepare subclass antiserums without contaminating antibodies to other IgG markers. Thus, we thought it worthwhile to standardize the antiserums with particular emphasis on specificity using several different technics. We found that some methods that have been used to define the subclass of red cell-bound IgG were inappropriate, and led to many false positive results.     

Robotics in the clinical laboratory

We are beginning to see the potential of robotics in the clinical laboratory through integration with automated analyzers and computer systems. However, there is a need for training programs that will prepare technologists to design and implement robotic systems for clinical laboratories. What will the robot laboratory of the future look like? We will see hospital laboratories begin to be located some distance away from the main facility because the labor component of staffing satellite laboratories will have been greatly reduced. Instrument manufacturers will see the need for analyzers that are robot-friendly and allow for simplified interfacing, both electronic and mechanical. Robots will become more versatile even to the point of performing complete instrument repair. Laboratories will be equipped with many task-oriented robotic stations, including, for example, accessioning and processing robots that prepare samples for transport by robotic carts. Analysis will be performed by a combination of robot and dedicated analyzer. Laboratory results will be reviewed by algorithms in the larger laboratory computer, which will alert the laboratory worker to unusual results. A large variety of analyses will be available to the patient with rapid turnaround. The end result will be more efficient health care delivery at reduced cost.     

Immunoblotting in the clinical laboratory

Since its introduction in 1979 protein blotting has become a routine tool in research laboratories. In the clinical laboratory its potential is becoming recognised for applications in fields such as infectious and autoimmune diseases, allergy and others. In this article we would like to outline the basic principles of protein blotting, to illustrate these with some examples related to clinical applications and to point out differences between these and classical methods.